Oligomers

ABSTRACT

Certain disclosed oligomers induce exon skipping during processing of myostatin pre-mRNA. The oligomers may be in a vector or encoded by the vector. The vector is used for inducing exon skipping during processing of myostatin pre-mRNA. A therapeutically effective amount of the oligomer may be administered to a subject patient such that exon skipping during processing of myostatin pre-mRNA is induced. The administration to a subject may be used in order to increase or maintain muscle mass, or slowing degeneration of muscle mass in the subject. The administration to a subject may ameliorate muscle wasting conditions, such as muscular dystrophy. Examples of such muscular dystrophies which may be so treated include Becker&#39;s muscular dystrophy, congenital muscular dystrophy, Duchenne muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy (FSHD), limb-girdle muscular dystrophy, myotonic muscular dystrophy, and oculopharyngeal muscular dystrophy.

FIELD OF THE INVENTION

The present invention relates to oligomers which are capable of causingexon skipping and, in particular, relates to oligomers which are capableof causing exon skipping in the myostatin gene.

BACKGROUND TO THE INVENTION

A range of strategies have been proposed to enhance muscle bulk andstrength as a treatment for a number of age-related muscle disorders andvarious neuromuscular disorders, including muscular dystrophies.Myostatin, a transforming growth factor-β family member, also calledgrowth and differentiation factor-8, is a negative regulator of musclegrowth and the myostatin signalling axis has been a major focus in suchstrategies. Myostatin null or hypomorphic animals are significantlylarger than wild-type animals and show a large increase in skeletalmuscle mass.¹ The first natural myostatin mutation in humans has alsobeen identified in a young boy.² Myostatin blockade, therefore, offers astrategy for counteracting muscle-wasting conditions including Duchennemuscular dystrophy.³ Delivery of myostatin-inhibiting genes, includinggrowth and differentiation factor-associated serum protein-1 (GASP-1),follistatin-related gene (FLRG), follistatin-344 (FS) and myostatinpropeptide, via adeno-associated virus,⁴⁻⁶ lead to an increase in musclemass in treated animals, with the greatest increase in FS-treatedanimals.⁷ Use of potentially therapeutic antimyostatin-blockingantibodies of high-binding affinity has proved to be a promisingstrategy. However, there are some constraints related to the use ofantimyostatin antibodies that include difficulty in long-termsustainability, undesirable immune responses, and inhibitory effects notprecisely specific to myostatin in regard to muscle growth.^(8,9)Significant increase in skeletal muscle mass was also observed usingadeno-associated virus vectors to deliver a recombinant myostatinpropeptide gene fragment, or by a retrovirus-based RNA interferencesystem (RNAi).^(4,6,10) Both approaches have safety concerns of possiblegenotoxicity, due to uncontrolled vector genome insertion into hostchromosomes.¹¹ The RNAi system faces an additional hurdle in terms ofeffective delivery of the RNAi molecules into the disease models forclinical studies.¹² RNA-based modulation therapy has the potential toovercome difficulties encountered by conventional gene therapy methods.Antisense oligonucleotides (AOs) are capable of hybridizing to a sensetarget sequence leading to cleavage of the RNA:DNA hybrid by RNase Hwhich results in downregulation of gene transcription.^(13,14) In analternative approach, antisense-mediated modulation of pre-mRNA splicinghas been pioneered by Dominski and Kole.¹⁵ In the first experiments, AOswere aimed at activated cryptic splice sites in the β-globin (HBB) andcystic fibrosis transmembrane conductance regulator (CFTR) genes inorder to restore normal splicing in β-thalassemia and cystic fibrosispatients.¹⁵⁻¹⁷ The identification of exon/intron boundaries by thesplicing machinery, and therefore inclusion of the exons into the mRNA,is extensively thought to depend on exonic splicing enhancer (ESE)motifs.¹⁸ By masking these ESE sites with sequence-optimized AOs, thetargeted exons are no longer recognized as exons, and are spliced outwith neighbouring introns. This so-called antisense-induced exonskipping has already been used clinically to partly correct the mutateddystrophin and convert the severe Duchenne muscular dystrophy phenotypeinto a milder Becker muscular dystrophy phenotype.¹⁹ Clinical trials todetermine the safety profile and the efficacy of single intramusculardoses of two different chemistries of AOs, 2′-O-methyl phosphorothioate(2′OMePS) AOs and phosphorodiamidate morpholino oligomers (PMOs) inDuchenne muscular dystrophy patients have recently beencompleted.^(20,21) The treatments were well tolerated by all thepatients and the injection of AOs induced the production of dystrophin.2′OMePS AOs, being negatively charged, are easily delivered in vitro,whereas PMOs are capable of more sustained effect in vivo due to theirresistance to enzymatic degradation²² and owing to their longersequence, have increased affinity to target.²³ When conjugated withvarious peptide derivatives, or with dendrimeric octa-guanidine(so-called Vivo-morpholino), PMOs demonstrate a significantly increaseddelivery in the case of dystrophin skipping.^(24,25)

SUMMARY OF THE INVENTION

The inventors have adopted the approach of using AOs with differentchemistries, so enhancing their half-lives relative to RNAi molecules,to investigate the outcome of myostatin knockdown by exon skipping.Skipping of exon 2 (374 nucleotides) of myostatin leads to anout-of-phase splicing of exons 1 and 3, and knockdown of myostatin dueto truncation of the Open Reading Frame and nonsense-mediated mRNAdecay. The data present here constitute a proof-of-principle thatoligonucleotide-mediated antisense exon skipping leads to aphysiologically significant myostatin knockdown in vitro and in vivo.This type of antisense treatment could thus form part of an effectivestrategy to improve various muscle-wasting conditions, and along withdystrophin rescue or augmentation, to treat Duchenne muscular dystrophy.

The present invention relates to oligomers which can bind to pre-mRNAproduced from the myostatin gene and cause exon skipping during cellularprocessing of the pre-mRNA.

The present invention provides an oligomer for inducing exon skippingduring processing of myostatin pre-mRNA, the oligomer comprising atleast 20 contiguous bases of a base sequence selected from the groupconsisting of:

(SEQ ID NO. 1)  1) XCXCGACGGGXCXCAAAXAXAXCCAXAGXX; (SEQ ID NO. 2)  2)XGXACCGXCXXXCAXAGGXXXGAXGAGXCX; (SEQ ID NO. 3)  3)CCXGGGXXCAXGXCAAGXXXCAGAGAXCGG; (SEQ ID NO. 4)  4)CAGCCCAXCXXCXCCXGGXCCXGGGAAGGX; (SEQ ID NO. 5)  5)XCXXGACGGGXCXGAGAXAXAXCCACAGXX; (SEQ ID NO. 6)  6)XGXACCGXCXXXCAXGGGXXXGAXGAGXCX; (SEQ ID NO. 7)  7)CCXGGGCXCAXGXCAAGXXXCAGAGAXCGG; (SEQ ID NO. 8)  8) XCCACAGXXGGGCXXXXACX;(SEQ ID NO. 9)  9) XCXGAGAXAXAXCCACAGXX; (SEQ ID NO. 10) 10)XCXXGACGGGXCXGAGAXAX; (SEQ ID NO. 11) 11) XGAXGAGXCXCAGGAXXXGC;(SEQ ID NO. 12) 12) XXCAXGGGXXXGAXGAGXCX; (SEQ ID NO. 13) 13)XXGXACCGXCXXXCAXGGGX; (SEQ ID NO. 14) 14) CAGAGAXCGGAXXCCAGXAX;(SEQ ID NO. 15) 15) XGXCAAGXXXCAGAGAXCGG; (SEQ ID NO. 16) 16)CCXGGGCXCAXGXCAAGXXX; (SEQ ID NO. 17) 17) CXGGGAAGGXXACAGCAAGA;(SEQ ID NO. 18) 18) XCXCCXGGXCCXGGGAAGGX; and (SEQ ID NO. 19) 19)CAGCCCAXCXXCXCCXGGXC,wherein X is T or U and the oligomer's base sequence can vary from theabove sequence at up to two base positions, and wherein the oligomer canbind to a target site in the myostatin pre-mRNA to cause exon skipping.

The oligomers described above cause exon skipping in the myostatin gene.In particular, these oligomers cause exon skipping of exon two of themyostatin gene, i.e. when the myostatin pre-mRNA is processed into mRNA,the oligomers stop exon two from being included in the mRNA.

Without being restricted to any particular theory, it is thought thatthe binding of the oligomers to the myostatin pre-mRNA interacts with orinterferes with the binding of SR proteins to the exon. SR proteins areinvolved in the splicing process of adjacent exons. Therefore, it isthought that interacting or interfering with the binding of the SRproteins interferes with the splicing machinery resulting in exonskipping.

The base “X” in the above base sequences is defined as being thymine (T)or uracil (U). The presence of either base in the sequence will stillallow the oligomer to bind to the pre-mRNA of the myostatin gene as itis a complementary sequence. Therefore, the presence of either base inthe oligomer will cause exon skipping. The base sequence of the oligomermay contain all thymines, all uracils or a combination of the two. Onefactor that can determine whether X is T or U is the chemistry used toproduce the oligomer. For example, if the oligomer is aphosphorodiamidate morpholino oligonucleotide (PMO), X will be T as thisbase is used when producing PMOs. Alternatively, if the oligomer is aphosphorothioate-linked 2′-O-methyl oligonucleotide (2′OMePS), X will beU as this base is used when producing 2′OMePSs. Preferably, the base “X”is only thymine (T).

The advantage provided by the oligomer is that it causes exon skipping.Preferably, the oligomer causes an exon skipping rate of at least 40%,i.e. exon two will be skipped 40% of the time. More preferably, theoligomer causes an exon skipping rate of at least 50%, more preferablystill, at least 60%, even more preferably, at least 70%, more preferablystill, at least 75%, more preferably, at least 80%, even morepreferably, at least 85%, more preferably still, at least 90%, even mostpreferably, at least 95%, more preferably, at least 98% and even morepreferably, at least about 99%. Exon skipping can be measured bytransfection (leashed or unleashed: concentration between 50 and 500 nM)into cultured human myoblast cells (e.g., using a transfection reagentsuch as Lipofectamine2000™), and evaluation of skipped and unskippedmRNAs by electrophoretic densitometric analysis of RTPCR reactionproducts.

The oligomer can be any type of oligomer as long as it has the selectedbase sequence and can bind to a target site of the myostatin pre-mRNA tocause exon skipping. For example, the oligomer can be anoligodeoxyribonucleotide, an oligoribonucleotide, a phosphorodiamidatemorpholino oligonucleotide (PMO) or a phosphorothioate-linked2′-O-methyl oligonucleotide (2′OMePS). Preferably, the oligomer is a PMOor a 2′OMePS. In one embodiment, the oligomer is a PMO. The advantage ofa PMO is that it has excellent safety profiles and appears to havelonger lasting effects in vivo compared to 2′OMePS oligonucleotides.Preferably, the oligomer is isolated so that it is free from othercompounds or contaminants.

The base sequence of the oligomer can vary from the selected sequence atup to two base positions. If the base sequence does vary at twopositions, the oligomer will still be able to bind to the myostatinpre-mRNA to cause exon skipping. Preferably, the base sequence of theoligomer varies from the selected sequence at up to one base positionand, more preferably, the base sequence does not vary from the selectedsequence. The less that the base sequence of the oligomer varies fromthe selected sequence, the more efficiently it binds to the target sitein order to cause exon skipping.

The oligomer is at least 20 bases in length. Preferably, the oligomer isat least 25 bases in length. In some embodiments, the oligomer may be atleast 28 bases in length or at least 30 bases in length. Preferably, theoligomer is no more than 40 bases in length. In some embodiments, theoligomer may be no more than 35 bases in length or no more than 32 basesin length. Preferably, the oligomer is between 20 and 40 bases inlength. More preferably, the oligomer is between 25 and 35 bases inlength. In some embodiments, the oligomer is between 28 and 32 bases inlength, between 29 and 31 bases in length, or about 30 bases in length.It has been found that an oligomer which is 30 bases in length causesefficient exon skipping. If the oligomer is longer than 40 bases inlength, the specificity of the binding to the target site may bereduced. If the oligomer is less than 20 bases in length, the exonskipping efficiency may be reduced.

In some embodiments, the oligomer comprises at least 20 contiguous basesof a base sequence selected from the group consisting of SEQ ID NOS.1-7, wherein the oligomer's base sequence can vary from SEQ ID NOS. 1-7at up to two base positions.

In other embodiments, the oligomer comprises at least 20 contiguousbases of a base sequence selected from the group consisting of SEQ IDNOS. 1-4, wherein the oligomer's base sequence can vary from SEQ ID NOS.1-4 at up to two base positions.

In the above embodiments relating to SEQ ID NOS. 1-7 and SEQ ID NOS.1-4, the oligomer preferably comprises at least 25 contiguous bases ofthe base sequences. More preferably, the oligomer comprises at least 28contiguous bases of the base sequences. In some embodiments, theoligomer comprises 30 contiguous bases of the base sequences, i.e. theoligomer comprises the base sequences of SEQ ID NOS. 1-7 or SEQ ID NOS.1-4. In the embodiments described in this paragraph, the oligomer's basesequence can still vary from SEQ ID NOS. 1-7 or SEQ ID NOS. 1-4 at up totwo base positions.

The oligomer may be conjugated to or complexed with various distinctchemical entities. For example, the oligomer may be conjugated to orcomplexed with a targeting protein in order to target the oligomer to,for example, muscle tissue. If the oligomer is conjugated to an entity,it may be conjugated directly or via a linker. In one embodiment, aplurality of oligomers may be conjugated to or complexed with a singleentity. For example, the oligomer may be conjugated to octa-guanidinedendrimers. Alternatively, an arginine-rich cell penetrating peptide(CPP) can be conjugated to or complexed with the oligomer. Inparticular, (R-Ahx-R)₄AhxB can be used, where Ahx is 6-aminohexanoicacid and B is beta-alanine (Moulton H M et al. (2007) Biochem. Soc.Trans. 35: 826-8.), or alternatively (RXRRBR)₂XB can be used²⁶ Theseentities have been complexed to known dystrophin exon-skipping oligomerswhich have shown sustained skipping of dystrophin exons in vitro and invivo.

Alternatively, a range of nanoparticle systems can be used to deliverthe oligomers⁷⁴.

In another aspect, the present invention provides a vector for inducingexon skipping during processing of myostatin pre-mRNA, the vectorencoding an oligomer of the invention, wherein when the vector isintroduced into a cell (e.g. a human cell), the oligomer is expressed.For example, it is possible to express antisense sequences in the formof a gene, which can thus be delivered on a vector. One way to do thiswould be to modify the sequence of a U7 snRNA gene to include anantisense sequence according to the invention. The U7 gene, completewith its own promoter sequences, can be delivered on an adeno-associatedvirus (AAV) vector, to induce bodywide exon skipping. Similar methods toachieve exon skipping, by using a vector encoding an oligomer of theinvention, would be apparent to one skilled in the art.

The present invention also provides a pharmaceutical composition forinducing exon skipping during processing of myostatin pre-mRNA, thecomposition comprising an oligomer as described above or a vector asdescribed above and a pharmaceutically acceptable carrier, adjuvant orvehicle.

Pharmaceutical compositions of this invention comprise an oligomer ofthe present invention, and pharmaceutically acceptable salts, esters,salts of such esters, or any other compound which, upon administrationto a subject (e.g. a human), is capable of providing (directly orindirectly) the biologically active oligomer thereof, with apharmaceutically acceptable carrier, adjuvant or vehicle.Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions of this invention include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, serum proteins, such as human serum albumin, buffer substancessuch as phosphates, glycine, sorbic acid, potassium sorbate, partialglyceride mixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

The pharmaceutical compositions of this invention may be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally, intradermally or via an implanted reservoir. Oraladministration or administration by injection is preferred. Thepharmaceutical compositions of this invention may contain anyconventional non-toxic pharmaceutically-acceptable carriers, adjuvantsor vehicles. The term parenteral as used herein includes subcutaneous,intracutaneous, intravenous, intramuscular, intra-articular,intrasynovial, intrasternal, intrathecal, intralesional and intracranialinjection or infusion techniques. Preferably, the route ofadministration is by injection, more preferably, the route ofadministration is intramuscular, intravenous or subcutaneous injectionand most preferably, the route of administration is intravenous orintramuscular injection.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluent,dispersant or similar alcohol.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, and aqueous suspensions and solutions. Inthe case of tablets for oral use, carriers which are commonly usedinclude lactose and corn starch. Lubricating agents, such as magnesiumstearate, are also typically added. For oral administration in a capsuleform, useful diluents include lactose and dried corn starch. Whenaqueous suspensions are administered orally, the active ingredient iscombined with emulsifying and suspending agents. If desired, certainsweetening and/or flavouring and/or colouring agents may be added.

The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of thisinvention with a suitable non-irritating excipient which is solid atroom temperature but liquid at the rectal temperature and therefore willmelt in the rectum to release the active components. Such materialsinclude, but are not limited to, cocoa butter, beeswax and polyethyleneglycols.

Topical administration of the pharmaceutical compositions of thisinvention is especially useful when the desired treatment involves areasor organs readily accessible by topical application. For applicationtopically to the skin, the pharmaceutical composition should beformulated with a suitable ointment containing the active componentssuspended or dissolved in a carrier. Carriers for topical administrationof the compounds of this invention include, but are not limited to,mineral oil, liquid petroleum, white petroleum, propylene glycol,polyoxyethylene polyoxypropylene compound, emulsifying wax and water.Alternatively, the pharmaceutical composition can be formulated with asuitable lotion or cream containing the active compound suspended ordissolved in a carrier. Suitable carriers include, but are not limitedto, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esterswax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Thepharmaceutical compositions of this invention may also be topicallyapplied to the lower intestinal tract by rectal suppository formulationor in a suitable enema formulation. Topically-transdermal patches arealso included in this invention.

The pharmaceutical compositions of this invention may be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

The pharmaceutical composition of the invention may also comprise anadditional biologically active agent. For example, where the compositionis for ameliorating Duchenne muscular dystrophy, the composition maycomprise an oligomer for causing exon skipping in the dystrophin gene.Such oligomers are described, for example, in U.S. application Ser. No.12/556,626.

The oligomers of the invention are for use in therapy and, inparticular, for use in inducing exon skipping during processing ofmyostatin pre-mRNA.

The present invention also provides a method of inducing exon skippingduring processing of myostatin pre-mRNA in a patient (e.g. a humanpatient), the method comprising administering a therapeuticallyeffective amount of the oligomer of the invention or the vector of theinvention to the patient such that exon skipping during processing ofmyostatin pre-mRNA is induced.

In the above method, the oligomers can be used to increase or maintainmuscle mass, or to slow the degeneration of muscle mass. In particular,musculoskeletal muscle mass can be increased or maintained, or itsdegeneration slowed. For example, the method can be for amelioratingmuscle wasting conditions such as degenerative muscular disorders,including various forms of muscular dystrophy. Degenerative musculardisorders such as various forms of muscular dystrophies can actually befatal at an early age of mid to late twenties. Muscle wasting conditionsthat can be ameliorated using the oligomers include muscular dystrophysuch as Becker's muscular dystrophy, congenital muscular dystrophy,Duchenne muscular dystrophy, distal muscular dystrophy, Emery-Dreifussmuscular dystrophy, facioscapulohumeral muscular dystrophy (FSHD),limb-girdle muscular dystrophy, myotonic muscular dystrophy andoculopharyngeal muscular dystrophy. In one embodiment, the method is forameliorating Duchenne muscular dystrophy (DMD). Other conditions thatcould be ameliorated by myostatin oligomers include cachexia (muscleloss due to for example, cancer, chronic obstructive pulmonary disease(COPMD) and HIV/AIDS), sarcopenia (muscle loss due to natural old age),muscle atrophy (muscle loss in denervating conditions such asmotorneuron disease, and spinal muscular atrophy). In addition,myostatin knockdown by oligomers and the ensuing increased muscle bulkmay also have the potential to counteract insulin-resistance in diabetesand obesity-related metabolic syndromes.

In some embodiments of the invention, another biologically active agentmay also be administered in a therapeutically effective amount. Forexample, where the method is for ameliorating DMD, antisense oligomersfor causing exon skipping in the dystrophin gene may also beadministered. Such oligomers are described, for example, in U.S.application Ser. No. 12/556,626.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will now be described in detail, by way of exampleonly, with reference to the accompanying figures in which:

FIGS. 1A-1B: Bioinformatics analysis, design, and evaluation in C2C12muscle cells of specific AOs predicted to induce skipping of myostatinexon 2. (FIG. 1A) Results from three algorithms used to identify ESEsequences for designing of exon skipping AOs targeting exon 2 of themouse myostatin gene. The ESE Finder analysis shows the location andvalues above threshold for SR protein-binding motifs, SF2/ASF, SF2/ASF(BRCA 1), SC35, SRp40, and SRp55 which are shown as vertical bars abovethe sequence of exon 2. The Rescue ESE analysis shows the position ofpossible exonic splicing enhancer sites by black horizontal linesparallel to the sequence of exon 2. The PESX analysis shows the locationof ESEs as light gray horizontal lines, and exon splicing silencers(ESSs) as dark gray horizontal lines. The bold horizontal laddered blacklines represent the sequence of the 20-mer 2′OMePSs which were obtainedafter aligning the outputs from the three algorithms. (FIG. 1B)Comparison of efficacy of different 2′OMePS oligomers to induce skippingof exon 2 in myostatin mRNA from C2C12 cell cultures. RT-PCR wasperformed on 1 μg mRNA from C2C12 cells treated with 12 different2′OMePS oligomers at 250 nmol/l. Transfections were performed induplicates and the nested RT-PCR products were loaded on 1.2% agarosegel as follow: Tracks 1 and 2: oligomer A1; Tracks 3 and 4: oligomer A2;Tracks 5 and 6: oligomer A3; Tracks 9 and 10: oligomer B1; Tracks 11 and12: oligomer B2; Tracks 13 and 14: oligomer B3; Tracks 15 and 16:oligomer C1; Tracks 17 and 18: oligomer C2; Tracks 19 and 20: oligomerC3; Tracks 23 and 24: oligomer D1; Tracks 25 and 26: oligomer D2; Tracks27 and 28: oligomer D3; Tracks 7, 8, 21 and 22: controls withtransfection reagent Lipofectamine 2000 alone, but no AO: Size Markerused is Hyper ladder IV. 2′OMePS, 2′O-methyl phosphorothioate RNA; AO,antisense oligonucleotide; bp, base pairs; ESE, exonic splicingenhancer; RT-PCR, reverse transcriptase-PCR.

FIG. 2: Antisense-induced myostatin exon 2 skipping with 2′OMePSoligomers leads to an increase in C2C12 cell proliferation. C2C12 cellswere treated with a range of 2′OMePS oligomers along with lipofectamine2000 (LF2000), and assayed 24 hours later for cell proliferation bylactic dehydrogenase assay. Treatment with 2′OMePS oligomers A3, B3, andD3 resulted in significant increases in the number of cells after 24hours compared to the cultures treated with only transfection reagentLF2000 but no AO. C3 did not induce a substantial change. (t-testanalysis, n=6; *P<0.05; **P<0.01). 2′OMePS, 2′O-methyl phosphorothioateRNA.

FIG. 3: Exon skipping in mouse following intramuscular injection of2′OMePS oligomers targeting myostatin exon 2. Oligomers A2 and B3 (3nmol) were administered by a single intramuscular injection into thetibialis anterior (TA) muscles of mice. Two and four weeks later,muscles were recovered, and RNA extracted and analyzed for the presenceof myostatin exon 2 skipping by RT-PCR. Agarose ethidium bromide gelelectrophoresis is shown for the products of RT-PCR analysis: The upperand lower bands correspond to the normal exon 1, 2, and 3 product (532bp) and the exon 2 skipped product (158 bp), respectively which wereverified by sequencing (data not shown). The faint shadow band ofintermediate migration in some tracks was found upon sequencing tocorrespond to a product containing a partial sequence of exon 2 due to acryptic 3′ splice site 296 nt downstream of the correct one. Tracks 1and 2: 14 days control; Tracks 3-5: 14 days A2-treated; Tracks 6-8: 28days A2-treated; Tracks 9-11; 14 days B3-treated; Tracks 12-14: 28 daysB3-treated; Tracks 15 and 16: 28 days control. Densitometric evaluationof the skipped and unskipped bands showed that after 14 days, A2 gave25.6% and B3 54.6% skipping, and after 28 days, A2 gave 48.6% and B324.5% skipping. 2′OMePS, 2′O-methyl phosphorothioate RNA; bps, basepairs; nt, nucleotides; RT-PCR, reverse transcriptase-PCR.

FIG. 4: Myostatin exon 2 skipping in C2C12 cell culture followingtreatment with a range of leashed-PMO lipoplexes. C2C12 cell culturestreated with a range of leashed PMOs in lipoplex form with LF2000exhibited skipping of exon 2 in myostatin mRNA. RT-PCR was performed on1 μg mRNA from C2C12 cells treated with 250 nmol/l PMOs (designed on thebasis of the most effective 2′OMePS sequences: A3, B3, C3, and D3) overa period of 24 hours. Transfections were performed in triplicate andRT-PCR products were loaded on 1.2% agarose gel as follows: Tracks 1-3:PMO-A; Tracks 4-6: PMO-B; Tracks 7-9: PMO C; Tracks 10-12: PMO-D; Tracks13-15: LF2000-treated control; bps, base pairs; LP2000, lipofectamine2000; PMO, phosphorodiamidate morpholino oligomers; RT-PCR, reversetranscriptase-PCR.

FIG. 5: Systemic injection of PMO conjugated to octa-guanidine dendrimer(Vivo-PMO) results in myostatin exon skipping associated with asignificant increase in muscle mass and myofiber size. Mice were treatedwith 6 mg/kg of Vivo-PMO-D3 by five weekly intravenous injections, andmuscles harvested for RNA extraction and immunohistology 10 days later.(a) Weight of soleus and EDL muscle after treatment. Weights of soleusmuscles were significantly increased (t-test, P<0.034; n=6) whereasweights of EDL muscles showed no significant change. (b) RT-PCR wascarried out on 1 μg RNA from soleus and EDL muscles and productsresolved on a 1.2% agarose gel. Track 1: Vivo-PMO treated soleus; Track2: control soleus; Track 3: Vivo-PMO treated EDL; Track 4: control EDL.(c) Distribution of myofiber sizes (CSA) in vivo-PMO treated (blackbars) and control (open bars) soleus muscles. (d) Representativedystrophin immunohistology indicating increased myofibre CSA in vivo-PMOtreated compared to control soleus muscle cryosections. Bar=500 μm. CSA,cross-sectional area; EDL, extensor digitorum longus; PMO,phosphorodiamidate morpholino oligomers; RT-PCR, reversetranscriptase-PCR.

INTRODUCTION

As stated above, the inventors have adopted the approach of using AOswith different chemistries, so enhancing their half-lives relative toRNAi molecules, to investigate the outcome of myostatin knockdown byexon skipping. Skipping of exon 2 (374 nucleotides) of myostatin waspredicted to lead to an out-of-phase splicing of exons 1 and 3, andknockdown of myostatin due to truncation of the Open Reading Frame andnonsense-mediated mRNA decay. The data present below constitute aproof-of principle that oligonucleotide-mediated antisense exon skippingleads to a physiologically significant myostatin knockdown in vitro andin vivo. This type of antisense treatment could thus potentially formpart of an effective strategy to improve various muscle-wastingconditions, and along with dystrophin rescue or augmentation, to treatDuchenne muscular dystrophy.

EXAMPLE 1

Materials and Methods

Bioinformatics Analysis of the Myostatin Gene to Design AOs Reagents.

Three different bioinformatics algorithms namely ESE Finder, PESX, andRescue ESE were used to design antisense reagents. Results from thethree algorithms were merged to define ESE sites and used to identifythe regions of the myostatin exon 2, which are expected to be optimaltargets for exon skipping antisense reagents. A set of 12 antisensereagents of 2′O-methyl RNA (2′OMePS) chemistry were designed to targetfour different ESE-rich regions of exon 2 of myostatin (FIG. 1A).

AO Reagents.

The 12 2′OMePS oligomers tested were obtained from Eurogentec (SA,Seraing, Belgium). The sequences of the 2′OMePS are as follows:

GDF8/A1: TCCACAGTTGGGCTTTTACT GDF8/A2: TCTGAGATATATCCACAGTT GDF8/A3:TCTTGACGGGTCTGAGATAT GDF8/B1: TGATGAGTCTCAGGATTTGC GDF8/B2:TTCATGGGTTTGATGAGTCT GDF8/B3: TTGTACCGTCTTTCATGGGT GDF8/C1:CAGAGATCGGATTCCAGTAT GDF8/C2: TGTCAAGTTTCAGAGATCGG GDF8/C3:CCTGGGCTCATGTCAAGTTT GDF8/D1: CTGGGAAGGTTACAGCAAGA GDF8/D2:TCTCCTGGTCCTGGGAAGGT GDF8/D3: CAGCCCATCTTCTCCTGGTC

PMOs were designed based on the 2′OMePS sequences. A total of four PMOswere tested and were obtained from Gene Tools (Philomath, Oreg.). PMOsequences are as follows:

Mstn A: TCTTGACGGGTCTGAGATATATCCACAGTT Mstn B:TGTACCGTCTTTCATGGGTTTGATGAGTCT Mstn C: CCTGGGCTCATGTCAAGTTTCAGAGATCGGMstn D: CAGCCCATCTTCTCCTGGTCCTGGGAAGGT

PMOs conjugated to octa-guanidine dendrimers (so-called Vivo-PMOs) werepurchased from Gene Tools.

Cell Culture and Transfection of C2C12 Cells with the Designed AntisenseReagents.

C2C12 mouse myoblasts were maintained in Dulbecco's modified Eagle'smedium (Sigma-Aldrich, Poole, UK) containing 10% fetal calf serum(Sigma-Aldrich), 4 mmol/l 1-glutamine, 100 U/ml penicillin and 100 μg/mlstreptomycin at 37° C. and 8% CO₂. Cells were split every 24 hours toprevent differentiation. Cells were detached by incubating them with0.15% trypsin-phosphate-buffered saline for 1 minute at 37° C., andseeded at a density of 1.5×105 cells/well of a 6-well plate. Theantisense reagents of 2′OMePS chemistry were transfected at 250 nmol/linto C2C12 cells using Lipofectamine 2000 (Invitrogen, Paisley, UK).Controls contained Lipofectamine 2000 but no antisense reagent. PMOswere leashed to complementary stretches of negatively charged DNA(obtained from MWG, Ebersberg, Germany) for efficient in vitrodelivery,²⁴ using Lipofectamine 2000 as transfection reagent. Alltransfections were performed in Dulbecco's modified Eagle's mediumcontaining 2 mmol/l glutamine (without serum and antibiotics) and after3-4 hours of transfection, the medium was replaced with full growthmedium containing serum as well as antibiotics. The transfections wereperformed in duplicate and the experiment repeated twice.

RT-PCR Analysis of Myostatin Exon Skipping.

For in vitro experiments, 24 hours after transfection, RNA was extractedfrom each well using QIAshredder/RNeasy extraction kit (Qiagen, Crawley,UK). For in vivo experiments, RNA was extracted from blocks using TRIzolreagent (Invitrogen, Scotland, UK). One microgram of RNA was reversetranscribed and resulting complementary DNA amplified using specificprimers obtained from MWG, using the Genescript kit (Genesys, Camberley,UK). One micro litre of PCR products obtained was used as a template fornested PCR. Sequences of the primers and details of the PCR protocolsused are available on request. The products from nested PCR wereseparated on 1.2% agarose gel in Tris-borate/EDTA buffer and HyperLadder IV (Bioline, London, UK) was used as the marker. Densitometricanalysis of the agarose gels was carried out using Gene Tools 3.05(Syngene, Cambridge, UK) and percentage skipping expressed as amount ofskipped product seen relative to total PCR products detected.

In Vitro Cell Proliferation Assay.

A proliferation assay using Cell Titer 96 Aqueous One Solution CellProliferation assay (Promega, Madison, Wis.) was performed, as reportedby Cory et al.,⁷⁵ on cells transfected with different 2′OMePS. Briefly,24 hours after seeding, the growth media was replaced with serum-freemedia and cells incubated at 37° C. After 24 hours of subjecting cellsto serum-free media, 15 μl of assay reagent was added to 75 μl cells ina 96-well plate. Plates were read at 490 nm. Statistical analysis on thedata from the proliferation assay was performed using the individualt-test.

Treatment of Mice with PMOs and Vivo-PMOs.

For all the in vivo experiments, animals (MF1 or C57B110) were boughtfrom Harlan (Blacktown, UK) and in-house maintained, and in vivoexperimentation conducted under statutory Home Office recommendation,regulatory, ethical and licensing procedures and under the Animals(Scientific Procedures) Act 1986 (project licence PPL 70/7008). Forintramuscular delivery, mice were anaesthetized and injected with 3 nmolof 2′OMePS (in 25 μl normal saline) into each of the TA muscles. Controlanimals were injected with normal saline. Whole body weights weremeasured weekly. TAs of treated and control mice were excised postmortemafter 2 weeks (n=4) and 4 weeks (n=4). Weights were measured and themuscles frozen in iso-pentane cooled with liquid nitrogen. For thesystemic administration, C57BL10 mice were injected intravenously with 6mg/kg of Vivo-PMO-D (Gene Tools) diluted in 200 μl of normal saline,every week for 5 weeks. Weights were measured weekly and various musclesfrom treated and control mice were harvested 10 days after the lastinjection. Cryosectioning was performed at 10 levels through the muscle.

Immunocytochemistry and Morphometry.

Hematoxylin and eosin staining was used to estimate the muscles size.For the estimation of fibre size and distribution, laminin staining wasperformed. Laminin antibody from Sigma-Aldrich (Dorset, UK) was used asprimary antibody, with biotinylated anti-rabbit immunoglobulin G (Dako,Glostrup, Denmark) as secondary antibody. Finally sections were stainedwith DAB (Vector Laboratories, Burlingame, Calif.) and slides mounted inDPX (VWR International, Poole, England) after appropriate washings.Immunostaining was also carried out with Dystrophin antibody. For this,H12 Polyclonal Rabbit antibody was used as primary antibody, and Alexafluor goat anti-rabbit 568 (fluorescein isothiocyanate) (Invitrogen,Paisley, Oreg.) was used as secondary antibody. Slides were mounted inVectashield mounting medium with DAPI (Vector Laboratories) afterappropriate washings with phosphate-buffered saline-Tween. CSA of musclefibres was measured using SigmaScan Pro 5.0.0 (Systat Software, London,UK).

Results

Bioinformatics Analysis and Design of Specific AOs Predicted to InduceSkipping of Myostatin Exon 2.

Bioinformatics analysis of exon 2 of myostatin was performed using threebioinformatics tools, ESE finder,^(27,28) PESX,^(29,30) and RescueESE,³¹ to identify and locate ESEs and exonic splicing suppressor orsilencer motifs. The output of these algorithms is displayed in FIG. 1A.A series of overlapping AOs were designed and synthesized as 2′OMePSsand PMOs to span sequences where clusters of ESEs which were predictedby one or more of the programs coincide.

High Levels of Myostatin Exon 2 Skipping in C2C12 Cell Culture FollowingTreatment with a Range of AOs.

In order to verify the efficiency of these AO target sequences, C2C12muscle cell cultures were transfected with the 2′OMePS oligomers, andnested reverse transcriptase-PCR (RT-PCR) for skipping of myostatinperformed on the RNA extracted from transfected and control cells. Arepresentative horizontal agarose gel electrophoresis separation ofproducts obtained is shown in FIG. 1B. The level of skipping produced byeach AO at 250 nmol/l was determined semiquantitatively usingdensitometric analysis.³¹ All of the designed 2′OMePSs were observed toinduce myostatin exon 2 skipping in C2C12 cultures but at various levelsof relative efficiency. A2 and A3 induced almost 100% skipping; B3(74%), C3 (41%), and D3 (48%) also induced a considerable level ofskipping. The nature of putative antisense-induced PCR exon1-exon3splicing product was confirmed by sequencing the products.

Antisense-Induced Myostatin Exon 2 Skipping and Myostatin KnockdownLeads to an Increase in C2C12 Cell Proliferation.

In order to verify that AO-mediated myostatin exon 2 skipping andknockdown, lead to a significant biological response, the autocrineactivity of myostatin on C2C12 cell proliferation was evaluatedfollowing treatment of cultures with 2′OMePSs targeting myostatin exon2. The cell proliferation assay was based on determination of lacticdehydrogenase activity of metabolically active cells. The results of theproliferation assay clearly showed a remarkable difference in cellproliferation in C2C12 cells treated with myostatin exon 2 AOs comparedto mock-transfected control cells (FIG. 2). Statistical analysis of thedata using individual paired t-tests showed that oligomers A3(P=0.0031), B3 (P=0.0055) and D3 (P=0.0115) induced a significantincrease in cell proliferation, as compared to mock transfected controlcells. Oligomer C3 (P=0.0534) did not produce a statisticallysignificant change.

Demonstration of Exon Skipping in Mouse Following IntramuscularInjection of 2′OMePS Oligomers Targeting Myostatin Exon 2.

On the basis of RT-PCR results obtained from the in-vitro studies, two2′OMePS oligomers (A2 and B3) were selected to evaluate their ability toinduce efficient exon skipping in vivo. The 2′OMePS oligomers (3 nmol)were administered by intramuscular injection into tibialis anterior (TA)muscles of mice. Two and four weeks after the injections, muscles wererecovered, weighed, RNA extracted and analyzed for the presence ofmyostatin exon 2 skipping by RT-PCR. Both reagents (A2 and B3) inducedsignificant level of myostatin exon 2 skipping at either the 2 weeks and4 weeks time points after a single 2′OMePS oligomer administration (FIG.3). Densitometric quantification of full-length and skipped productbands from the RT-PCR analyses of RNA was performed to detect which ofthe two 2′OMePSs tested was the more efficient in vivo. Oligomer A2 gave25.6% skipping, and B3 gave 54.6% skipping at the 2 weeks time point.However, after 4 weeks, A2 gave 48.6% skipping whereas B3 gave 24.5%skipping. Although the skipping of myostatin exon 2 was evident, theeffect was not sufficient to see a significant change in TA muscle mass(data not shown). From previous work on exon skipping for dystrophin, itis well established that the intramuscular injections of nakedunconjugated AOs in undamaged muscles are not very efficient.²⁵

High Levels of Myostatin Exon 2 Skipping in C2C12 Cell Culture FollowingTreatment with a Range of PMOs Designed on the Basis of 2′OMePS Data.

The animal studies above established that exon skipping of the myostatingene observed after intramuscular injection of 2′OMePS AOs wasinsufficient to induce change in TA mass. The PMO chemistry has beendemonstrated to have very high efficiency in vivo.³³ Therefore, PMOswere designed on the basis of most efficient 2′OMePS AOs (A3, B3, C3,and D3) and initially tested in vitro. PMOs are uncharged chemicals anddo not directly interact with the polycationic transfection reagentlipofectamine 2000. In order to enable reasonable transfectionefficiency in C2C12 cells, PMOs were hybridized to complementaryso-called leash oligonucleotides of natural negatively charged DNAchemistry as previously described.^(23,34,35) Nested RT-PCR analysis ofmRNA harvested from C2C12 cells treated with leashed-PMO lipoplexesdemonstrated that exon skipping was induced by all the PMOs accurateskipping of the targeted exon by both AO chemistries tested here.

Systemic Injection of PMOs Conjugated to Octa-Guanidine DendrimerResulted in Myostatin Exon Skipping Associated with a SignificantIncrease in Muscle Mass and Myofibre Size.

The conjugation of PMO with octa-guanidine dendrimer (so-calledVivo-PMOs) significantly increases the delivery and efficiency of PMOdirected against exon 23 of dystrophin compared to unmodified PMO.²⁵Therefore, a Vivo-PMO based on the sequence of the previously tested2′OMePS oligomer, D3, was produced to evaluate systemic intravasculartreatment regimes. Mice were treated with 6 mg/kg of Vivo-PMO-D3 by fiveweekly intravenous injections, and whole body weight and the mass of TA,soleus, and extensor digitorum longus (EDL) muscles were recorded 10days after the last injection. Among the muscles analyzed in the treatedanimals the soleus showed a statistically significant change in mass(P=0.034) (FIG. 5a ). In accordance with this, high levels of exonskipping of myostatin exon 2 was demonstrated at the transcript level insoleus (79%), whereas a very low level of skipping was observed in EDLmuscle (9%) (FIG. 5b ). Importantly, the cross-sectional area (CSA) ofsoleus muscle fibres in treated animals significantly increased(P<0.0001; mean CSA were 254±5 μm2 for control and 333±3 μm2 forPMO-treated animals (n=6) with a significant shift on the distributionof CSA (η2=38.34; df=12) (FIG. 5c, d ). No change was observed in theCSA of EDL muscle.

Discussion

Although targeting donor splice site, acceptor splice sites and branchpoint sequences has successfully led to exon exclusion including DMDexon skipping³⁶⁻³⁸, some studies have proved that targeting splice sitesdoes not always induce exon skipping and therefore exclusion of an exonfrom the pre-mRNA³⁹ These contain some consensus sequences common tomany other genes; therefore there lies a possible risk of disrupting thesplicing of non-specific genes⁴⁰. Exon splicing enhancers (ESEs) motifsform the binding sites for SR-protein RNA domains and thus help thesplicing machinery in exon recognition⁴¹. It has been shown thatintraexonic point mutations usually lead to mRNA level of exon skippinginstead of misense or no change in the amino acids⁴². As SR proteinbinding to ESEs is very crucial for exon exclusion, blocking the ESEswith Antisense oligonucleotides (AOs) would be expected to result inexon skipping. Different software like RESCUE ESE^(43,44), ESEFinder⁴⁵and PESX⁴⁶ have been widely used to predict possible ESE sites fordifferent SR domains in order to assist in designing AOs^(40,47,48).

Different oligonucleotide sequences of two different chemistries totarget myostatin exon 2 were designed using these available online toolswhich showed a promising level of exon skipping. 2′OMePS chemistry wasused for the preliminary tissue culture studies because of the advantageof cheap and easy synthesis over some other the PMO chemistry^(49,50).RNA from all the C2C12 cells transfected with twelve different 2′OMePSAO sequences showed skipped myostatin exon 2 as demonstrated by RT-PCRalong with the full length product. As myostatin has been established tobe a negative regulator of muscle mass growth and differentiation⁵¹⁻⁵³,a decrease in its level is expected to result in enhanced proliferativecapacity of muscle cells^(54,55). Therefore, a colorimetricproliferation assay based on the principle of bioreduction of atetrazolium compound by viable cells gives a quantitative measure ofliving cells present in a system⁵⁶. On performing this assay on cellstreated with four different AOs (one from each of the four sets based onRT PCR results), it was observed that the cells treated with AO-A3showed increased proliferation compared to control cells (p<0.01),treatment with AOs B3 and D3 also showed an increased cell proliferation(p<0.05), whereas AO-C3 did not lead to a significant increase in levelof proliferation compared to the control cells.

PMO chemistry has high nuclease resistance⁵⁷ and it does not induceRNase H-mediated down-regulation of the mRNA that it targets⁵⁸. Due touncharged background, however, PMOs cannot be delivered efficientlyacross cells using cationic liposomes and needs to be used at very highconcentrations^(59,60). Therefore, an anionic single-stranded nucleicacid molecule called ‘leash’ was annealed to the PMO in order to mediatecomplex formation of PMO with the cationic transfection reagent⁶¹. Fourdifferent 20-mer 2′OMePS AO sequences were used for design and synthesisof PMOs. PMOs were 30-mers with an overlap of 10 bases between AO-2 andAO-3 of each of the 2′OMePS AO sets, A(1,2,3), B(1,2,3), C(1,2,3) andD(1,2,3). All the PMOs linked to their respective leashes resulted ininduction of exon 2 skipping of myostatin mRNA and therefore showed thefeasibility of the approach with two different AO chemistries.

A luciferase reporter assay has been used to study the myostatininhibition effect of myostatin propeptide as well as that of myostatinneutralizing antibody JA16 in terms of a decrease in Smad binding to aTGF-β responsive elements called CAGA boxes⁶²⁻⁶⁴. A dose-response curvewas prepared using different dilutions of recombinant mouse myostatinusing human rhabdomyosarcoma cell line, A204 transfected withpGL3-(CAGA)₁₂-luc. When supernatant from C2C12 cells treated with PMO-Dwas assayed on the A204 cells transfected with luciferase reportercontaining Smad-binding site (CAGA) and luminescence was recorded, therewas found to be a significant decrease (p=0.011) in the relative lightunits (RLU) after 48 hours in case of supernatant from treated C2C12cells compared to supernatant from control C2C12 cells. This indicates adecrease in the transcriptional activity of endogenous Smad proteinswhich are crucial for TGF-β-mediated signal transduction⁶² Therefore,inhibition of myostatin by exon skipping results in reduced biologicalactivity related to modulation of myostatin pathway. This study thusconfirms the reported results for myostatin blockade using dominantnegative ActRIIB in human myoblasts⁶⁵ myostatin-neutralizing antibody,JA16⁶⁴ and myostatin propeptide⁶³ showing a decrease in Smad2transcriptional activity and thus antagonizing biological activity. Whenthe mean RLU values from the reporter assay for PMO-D treated cells wereplotted on the myostatin standard curve, there was found to be adecrease in myostatin concentration in treated samples relative to thecontrol cells by 44%. All these results were evident of skipping ofmyostatin exon 2 in vitro using two different chemistries along withmodulation of proliferative capacity as well as alteration of myostatinpathway of AO-treated cells.

Further Discussion

It has been well established that myostatin is a negative regulator ofskeletal muscle mass⁶⁶ and several approaches have been used toknockdown this factor to induce an increase in skeletal muscle growth.⁶⁷The use of AOs to induce exon skipping and thereby knockdown theexpression of myostatin presents several advantages over the othercurrently used gene therapy approaches. Firstly, there is no risk ofuncontrolled insertion into the genome with AOs as in case ofvirus-mediated approaches.⁶⁸ Moreover, with an appropriate dosingregimen, exon skipping levels can be regulated and, if necessary thetreatment can be interrupted. Importantly AOs have not been reported toproduce any toxic effects or immune response so far in animal models aswell as when used in clinical application.⁶⁹ Here, the inventors showthat AOs of 2′OMePS chemistry, designed using bioinformatics algorithms,resulted in a substantial level of myostatin exon 2 skipping in vitro.Myostatin being an inhibitor of myogenic differentiation, controls theproliferation of myoblasts.⁷⁰ Therefore, myostatin knockdown is expectedto increase the cell proliferation. The AOs designed were biologicallyactive and induced an increase in C2C12 cell proliferation. The efficacyof knockdown by exon skipping in vivo has proved to be more challengingto establish than in vitro. The efficiency of myostatin skipping wasverified by injecting 2′OMePS intramuscularly. The intramusculartreatment of a single muscle induced exon skipping, but did not appearto affect myostatin activity. This is likely to be due to supply ofbiologically active myostatin to the injected muscle by the bloodstream.Moreover, in a hypothetical clinical approach, the whole body should betreated. For these reasons, the inventors decided to administer the AOsby systemic tail vein injection in further experiments. PMO chemistrywas chosen for this experiment due to its better stability compared tothe 2′OMePS and also because PMOs have been reported to have a longereffect in vivo.⁷¹ This is particularly important for knocking downproteins like myostatin, which do not have a long half-life likedystrophin. The PMO sequence used for the systemic administration studyinduced efficient skipping in vitro. It also maps in a region totallyconserved between mouse and human myostatin paving the way to test thesame PMO for clinical applications in humans. In order to achieve areasonable effect in undamaged muscles, a PMO conjugated to a deliverymoiety has to be used.⁷² Vivo-PMO is commercially available and has beenreported to be effective in normal healthy mice.²⁵ By injectingVivo-PMO, a substantial increase in muscle size and the change of CSAfibre distribution has been obtained, but only in the soleus muscle. Thedifferential response in EDL and soleus may be due in part to a greateramount of ActRIIb being expressed on the surface of EDL muscle, orbecause the intrinsic level of myostatin is greater in fast (myosin typeIIb positive) myofibres.^(4,6) Alternatively, it can be speculated thatthe dosing regimen used, which has been reported to be optimal forVivo-PMO for exon skipping of dystrophin gene,²⁵ does not achievesufficient skipping of myostatin gene in EDL. In the case of dystrophinskipping, the half-life of dystrophin protein and mRNA is extremely longand therefore relatively smaller dosage of AO gives more sustained exonskipping.⁷³ However, in myostatin skipping, it is perhaps likely thatmore frequent redosing is required, to have a more sustained presence ofAOs. This may explain the transient and weak effect in terms of wholebody weight change that was observed in vivo. Interestingly, only soleusmuscle showed a significant increase in weight and CSA fibredistribution. This is in compliance with some previously published datashowing that soleus is the most affected muscle following a systemicapproach to knockdown myostatin.⁴ The results represent aproof-of-principle that myostatin knockdown can be obtained by skippingan exon from the transcript by using AOs.

EXAMPLE 2

The 30-mer PMO AOs tested above (Mstn-A to Mstn-D) were designed totarget the myostatin gene in mice. Therefore, these Mstn-A to Mstn-Dsequences correspond (are complementary to) to the Genbank mousemyostatin cDNA/mRNA gene sequence. The corresponding sequencescomplementary to the Genbank human myostatin sequences are as followswith differences between the Genbank mouse and human underlined:

Hum Mstn A: TCTCGACGGGTCTCAAATATATCCATAGTT Hum Mstn B:TGTACCGTCTTTCATAGGTTTGATGAGTCT Hum Mstn C:CCTGGGTTCATGTCAAGTTTCAGAGATCGG Hum Mstn D:CAGCCCATCTTCTCCTGGTCCTGGGAAGGT

The skipping efficiency of these AOs can be tested by transfection(leashed or unleashed: concentration between 50 and 500 nM) intocultured human myoblast cells (eg using a transfection reagent such asLipofectamine2000™), and evaluation of skipped and unskipped mRNAs byelectrophoretic densitometric analysis of RTPCR reaction products.

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The invention claimed is:
 1. An oligomer of 20-40 bases comprising atleast 20 contiguous bases of: 1) XCXCGACGGGXCXCAAAXAXAXCCAXAGXX (SEQ IDNO. 1); or 2) a base sequence that varies from SEQ ID NO. 1 at up to twobase positions, wherein X is T or U, wherein the oligomer is aphosphorodiamidate morpholino oligomer, and wherein the oligomer bindsto a target site in the myostatin pre-mRNA to cause exon skipping. 2.The oligomer of claim 1, wherein the oligomer is about 30 bases inlength.
 3. The oligomer of claim 1, wherein the oligomer comprisesXCXCGACGGGXCXCAAAXAXAXCCAXAGXX (SEQ ID NO. 1).
 4. The oligomer of claim1, wherein the oligomer is of 30 bases comprisingXCXCGACGGGXCXCAAAXAXAXCCAXAGXX (SEQ ID NO. 1).
 5. The oligomer of claim1, wherein the oligomer comprises at least 25 contiguous bases ofXCXCGACGGGXCXCAAAXAXAXCCAXAGXX (SEQ ID NO. 1).
 6. The oligomer of claim1, wherein the oligomer is conjugated to or complexed with a distinctchemical entity.
 7. An expression vector encoding an oligomer of 20-40bases comprising at least 20 contiguous bases of: 1)XCXCGACGGGXCXCAAAXAXAXCCAXAGXX (SEQ ID NO. 1); or 2) a base sequencethat varies from SEQ ID NO. 1 at up to two base positions, wherein X isT or U, and wherein the oligomer binds to a target site in the myostatinpre-mRNA to cause exon skipping.
 8. A method of inducing exon skippingduring processing of myostatin pre-mRNA in a patient, the methodcomprising administering a therapeutically effective amount of theoligomer of claim 1 or the vector of claim 7 to the patient such thatexon skipping during processing of myostatin pre-mRNA is induced.
 9. Themethod of claim 8, wherein the method is for increasing or maintainmuscle mass, or slowing degeneration of muscle mass in the patient. 10.The method of claim 8, wherein the method is for ameliorating musclewasting conditions.
 11. The method of claim 8, wherein the method is forameliorating a muscular dystrophy such as Becker's muscular dystrophy,congenital muscular dystrophy, Duchenne muscular dystrophy, distalmuscular dystrophy, Emery-Dreifuss muscular dystrophy,facioscapulohumeral muscular dystrophy (FSHD), limb-girdle musculardystrophy, myotonic muscular dystrophy and oculopharyngeal musculardystrophy.
 12. The method of claim 8, wherein the method is forameliorating Duchenne muscular dystrophy.
 13. The method of claim 12,wherein the method further comprises administering a therapeuticallyeffective amount of an oligomer which causes exon skipping in thedystrophin gene and which ameliorates Duchenne muscular dystrophy. 14.An oligomer of 20-40 bases comprising at least 20 contiguous basesof: 1) XCXCGACGGGXCXCAAAXAXAXCCAXAGXX (SEQ ID NO. 1); or 2) a basesequence that varies from SEQ ID NO. 1 at up to two base positions,wherein X is T or U, wherein the oligomer is a phosphorothioate-linked2′-O-methyl oligomer, and wherein the oligomer binds to a target site inthe myostatin pre-mRNA to cause exon skipping.
 15. The oligomer of claim14, wherein the oligomer is about 30 bases in length.
 16. The oligomerof claim 14, wherein the oligomer comprisesXCXCGACGGGXCXCAAAXAXAXCCAXAGXX (SEQ ID NO. 1).
 17. The oligomer of claim14, wherein the oligomer is of 30 bases comprisingXCXCGACGGGXCXCAAAXAXAXCCAXAGXX (SEQ ID NO. 1).
 18. The oligomer of claim14, wherein the oligomer comprises at least 25contiguous bases ofXCXCGACGGGXCXCAAAXAXAXCCAXAGXX (SEQ ID NO. 1).
 19. The oligomer of claim14, wherein the oligomer is conjugated to or complexed with a distinctchemical entity.